There are numerous human and animal diseases or infections that can be caused by Gram-negative bacteria such as, for example, Bordetella spp. and Haemophilus ducreyi. 
Vaccination has proven effective for preventing infection of humans and animals by Bordetella spp. Killed whole cell and subunit vaccines have been used to immunize parenterally to protect humans against pertussis caused by Bordetella pertussis, a highly contagious, severe respiratory infection especially of young children. B. parapertussis causes a milder and less frequent form of the disease, but its incidence and importance is garnering increasing attention. No vaccine is known to prevent it. Vaccination against B. pertussis does not protect against B. parapertussis. Parapertussis infection followed by pertussis in the same individuals has been described in literature. B. pertussis is confined to human, while B. parapertussis is confined to human and sheep. B. bronchiseptica causes respiratory infections in a variety of hosts: kennel cough in dogs, atrophic rhinitis in piglets, bronchopneumonia in rabbits and guinea pigs. Rarely, it infects humans, but young children, animal handlers and increasingly immuno-compromised individuals are susceptible. Unlike most bacterial respiratory pathogens, B. bronchiseptica efficiently colonizes the ciliated epithelium of the respiratory tract of the host and may establish chronic infections. A cellular veterinary vaccine is available but it is of limited efficacy.
It has been shown that protection to the infections caused by gram-negative bacteria can be conferred by serum anti-lipopolysaccharide (LPS) IgG. Cohen et al., “Double-blind vaccine-controlled randomised efficacy trial of an investigational Shigella sonnei conjugate vaccine in young adults,” Lancet 349(9046):155-159, 1997. The LPSes of all three bordetellae share several similar features, though none of them is identical in structure. B. pertussis produce rough-type LPS comprising a Lipid A domain and branched dodecasaccharide chain, carrying unusual sugars and free amino and carboxylic groups. On the basis of SDS-PAGE migration, it is divided into Band B—Lipid A and a branched nanosaccharide, that if further substituted by a trisaccharide unit is termed Band A. Almost identical core structure was reported for B. bronchiseptica LPS. On the contrary, B. parapertussis core region has a simplified heptasaccharide structure; it does not contain Band A trisaccharide and Band B lacks one heptose and one N-acetylgalactosamine substituents. Only B. bronchiseptica and B. parapertussis synthesize O-specific polysaccharides (O-SP) and initially it was reported that they carry identical structure of linear polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galactouronic acid (Di Fabio J L et al., FEMS Microbiol. Lett 97:275-282, 1992). However later, serological differences between B. bronchiseptica strains were observed and ascribed to the structural variations of the non-reducing end-groups of LPS O-chains (Vinogradov E. et al., Eur. J. Biochem. 276:7230-7236, 2000). As it was reported for Vibrio cholerae O1 serotype Ogawa and Inaba, the non-reducing end-groups play a significant role as major epitopes in serological reactions (Wang J., J. Biol. Chem. 273:2777-2783, 1998). Similar observation was made in case of Salmonella O40 and O43 serotypes.
Chancroid is a sexually transmitted genital ulcer disease (GUD) caused by the bacterium Haemophilus ducreyi. Chancroid presents with characteristic and persistent genital ulcers on the external genitals, associated with regional lymphadenopathy in 50% of cases. The disease is common in many developing countries, and is considered a significant risk factor together with other genital GUD, e.g. herpes simplex virus 2 (HSV-2) for heterosexual HIV transmission in geographic areas where both diseases are prominent.
A number of putative virulence factors of H. ducreyi have been described which may play a role in pathogenicity of this organism. Two of these factors are toxins: a hemolytic toxin and cytolethal distending toxin. The outer membrane proteins, DsrA and DltA, have been shown to promote resistance to killing by normal human serum. The hemoglobin receptor HgbA and the Cu, Zn-superoxide dismutase both seem to play a role in iron acquisition for H. ducreyi. Filamentous hemagglutinin like protein is involved in inhibition of phagocytosis. Heat-shock proteins (HSP) of H. ducreyi situated on the surface of the bacteria are responsible for protection of these bacteria against changes in the environment and enhance H. ducreyi adhesion to mammalian cells. Additionally, a number of proteins have been shown to play a role in adherence.
The lipooligosaccharide (LOS) produced by H. ducreyi is a putative virulence facto, as well. Previous studies have shown that LOS plays a role in adherence of bacteria to keratinocytes and human foreskin fibroblasts and also contribute to the development of lesions in animal models. Structural studies have been performed on the LOS from a number of H. ducreyi strains, e.g. 35000, ITMA 2665, 3147, 5535, CCUG 7470, 4438 and others. These studies have shown that the predominant form of the core oligosaccharide of the LOS is composed of 10 saccharides with a lactosamine or sialyllactosamine at the non-reducing end and is expressed by majority of strains.
H. ducreyi enters the skin or mucosa through wounds and attaches to extracellular matrix and to cells. This stimulates an inflammatory response with the development of pro-inflammatory cytokines and assembly of phagocytic cells; granulocytes and macrophages, at the infection site. H. ducreyi may be found both intra and extracellularly. The inflammatory process may clear the organism partially but may also cause tissue destruction and chronic infection with granuloma formation as observed in rabbit model of infection.
The mediators of immunity to chancroid are not known. Data from patients and infected volunteers indicate that this local infection does not confer immunity against subsequent re-infection and do not induce an antibody response. The results from these experiments indicate that the cytokine pattern and the type of cells involved in the early immune response to H. ducreyi, may have features of a Th1 response, including a poor or no antibody response. The in vitro studies of interactions of H. ducreyi with human monocyte-derived-dendritic cells and with macrophages confirmed an initial Th1 response. Studies in a rabbit model showed that both antibodies and cellular immunity contributed to reducing the number of bacteria in the lesions, thus contributing to protection. Data from a swine model indicated that antibodies alone, at levels achieved only after more than 3 injections of live bacteria, are sufficient for protection. Antibodies to different bacterial cell components were detected in the late stage of disease in sera from patients with chancroid, but antibodies neutralizing CDT have been detected only in about 28% of chancroid cases. It has also been noted that antibodies specific to the LOS of this organism enhance opsonophagocytic killing of H. ducreyi in vitro, but such antibodies are not elicited in sufficient amounts after repeated dermal injections of bacteria to animals. Low level of induced LOS antibodies may be due to the fact that the LOS structure resembles terminal saccharides of paraglobise, a major antigen on human erythrocytes and muscles. Since re-infection with H. ducreyi can occur, the immunity, including the amount and specificity of antibodies elicited by this local infection, is likely not sufficient for protection.
The covalent binding of oligosaccharide to carrier proteins by random activation of the saccharide using CDAP and ADH as the linker, resulted in conjugates that induced higher levels of IgG anti LOS than repeated injections of the whole cell (Lundquist A, Ahlman k T. Lagergärd, “Preparation and immunological properties of Haemophilus ducreyi lipooligosaccharide-protein conjugates,” ASM Meeting, abstract e-044, New Orleans, 2004). A vaccine to prevent chancroid would reduce/prevent the disease burden and have the added benefit of reducing HIV incidence.
Shigellae are Gram-negative bacteria, pathogens to humans only, that can cause endemic and epidemic dysentery worldwide, especially in the developing countries. The symptoms usually start with watery diarrhea that later develops into dysentery, characterized by high fever, blood and mucus in the stool, and cramps. Shigella flexneri causes dysentery mostly in developing countries with more fatalities then any other Shigella species. The disease can be prevented by vaccination using the polysaccharide part of the LPS as an immunogen.